running title authors - biorxiv...2019/08/01 · ventura & harris, 1999). astrocytes are also...

Short and long TNF-alpha exposure recapitulates canonical astrogliosis

events in human induced pluripotent stem cells-derived astrocytes

Running title

Astrogliosis in human astrocytes

Authors

Pablo Trindade1,2; Erick Correia Loiola1; Juciano Gasparotto3; Camila

Tiefensee Ribeiro3; Pablo Leal Cardozo4; Sylvie Devalle1; José Alexandre

Salerno1; Isis Moraes Ornelas1; Pitia Flores Ledur 1; Fabiola Mara Ribeiro4;

Ana Lucia Marques Ventura5; José Claudio Fonseca Moreira3; Daniel Pens

Gelain3; Lisiane Oliveira Porciúncula3; Stevens Kastrup Rehen1,6

Affiliations

1. Instituto D’Or de Pesquisa e Ensino (IDOR), Rio de Janeiro, Brazil.

2. Pós-Graduação em Biologia Molecular e Celular, Universidade Federal do

Estado do Rio de Janeiro, Brazil.

3. Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde,

Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

4. Departamento de Bioquímica e Imunologia, Universidade Federal de

Minas Gerais, Belo Horizonte, Brazil.

5. Departamento de Neurobiologia, Universidade Federal Fluminense,

Niterói, Brazil.

6. Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro,

Rio de Janeiro, Brazil.

.CC-BY 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted August 1, 2019. . https://doi.org/10.1101/722017doi: bioRxiv preprint

https://doi.org/10.1101/722017

http://creativecommons.org/licenses/by/4.0/

2

Acknowledgements

We thank Fernanda Albuquerque, Gabriela Lopes Vitória, Ismael Carlos da

Silva Gomes, Jarek Sochacki, Marcelo do Nascimento Costa and Renata

Maciel Santos for technical support. The authors declare no competing

financial interests. This work was sponsored by Fundação de Amparo a

Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Instituto Nacional

de Neurociência Translacional (INNT) and Banco Nacional de

Desenvolvimento (BNDES), in addition to intramural grants from D’Or Institute

for Research and Education.

Word Count

Total: 7683; Title: 16; Running Title: 4; Authors: 45; Affiliations: 86

Acknowledgements: 90; Abstract: 247; Keywords: 10; Main Points: 31;

Introduction: 674; Material and Methods: 1708; Results: 1062; Discussion:

1053; References: 1696; Figure Legends: 846; Author Contributions: 137.

Abstract

Astrogliosis comprises a variety of changes in astrocytes that occur in a

context-specific manner, triggered by temporally diverse signaling events that

vary with the nature and severity of brain insults. However, most mechanisms

underlying astrogliosis were described using animal models, which fail to

reproduce some aspects of human astroglial signaling. Here, we report an in

vitro model to study astrogliosis using human induced pluripotent stem cells


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(iPSC)-derived astrocytes which replicates aspects temporally intertwined of

reactive astrocytes in vivo. We analyzed the time course of astrogliosis by

measuring nuclear translocation of NF-kB, secretion of cytokines and changes

in morphological phenotypes of human iPSC-derived astrocytes exposed to

TNF-α. It was observed the NF-kB nuclear translocation, increases either in

the inflammation-related cytokines secretion and gene expression for IL-1β,

IL-6 and TNF-α following 24 h TNF-α stimulation. After 5 days, human iPSC-

derived astrocytes exposed to TNF-α exhibited increases in vimentin and

GFAP immunolabeling, elongated shape and shrinkage of nuclei, which is

typical phenotypes of astrogliosis. Moreover, about a 50% decrease in D-[3H]

aspartate uptake was observed over the astrogliosis course with no evident

cell damage, which suggests astrocytic dysfunction. Taken together, our

results indicate that cultured human iPSC-derived astrocytes reproduce

canonical events associated to astrogliosis in a time dependent fashion. Our

findings may contribute to a better understanding of mechanisms governing

human astrogliosis. Furthermore, the approach described here presents a

potential applicability as a platform to uncover novel biomarkers and new drug

targets to refrain astrogliosis associated to human brain disorders.

Keywords

reactive astrocytes, neuroinflammation, induced pluripotent stem cells;

astrogliosis; cytokines; TNF-alpha


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1. Introduction

Astrocytes are the most abundant cells of the mammalian central nervous

system (CNS). The astrocytic processes enwrap both pre- and post-synaptic

elements and closely approach the synaptic cleft, thus modulating synaptic

transmission (Perez-Alvarez, Navarrete, Covelo, Martin, & Araque, 2014;

Ventura & Harris, 1999). Astrocytes are also known for their secretory

potential, releasing neurotransmitters, ions and other signaling molecules to

the extracellular milieu in order to coordinate synaptic homeostasis

(Benarroch, 2016; Verkhratsky, Matteoli, Parpura, Mothet, & Zorec, 2016).

As a cell population with heterogeneous morphology and functioning,

astrocytes from rodents and humans show distinct features regarding

morphology, gene expression and functional competencies (Zhang et al.,

2016). Roughly, the population of human cortical astrocytes is composed of

larger and morphologically more complex cells when compared to cortical

astroglia from rodents (Oberheim et al., 2009). Yet, multiple subclasses of

astrocytes described in the human neocortex are not found in the same region

of the murine brain (Oberheim et al., 2009).

Astrocytes are prompt to react after inflammatory insults, experiencing a

continuum of temporally-ordered physiological changes that culminates in a

process called astrogliosis. Initially, mechanical or pathological injuries in the

CNS trigger NF-kB signaling in astrocytes leading to an increased production

of NF-kB-dependent cytokines, which potentially activate these cells (Lattke et

al., 2017; Saggu et al., 2016). Once activated, astrocytes show progressive

upregulation of intermediate filaments GFAP and Vimentin (Liu et al., 2014)


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and heterogenous degrees of cell hypertrophy (Kang, Lee, Han, Choi, &

Song, 2014). As long as the reactive phenotype is maintained, astrocytes may

present impaired metabolic functions such as disrupted recycling of

neurotransmitters (Schreiner, Berlinger, Langer, Kafitz, & Rose, 2013) and

energy metabolism (Gavillet, Allaman, & Magistretti, 2008). These biological

events are intertwined and occur within a time course which determines the

extension of the lesion (Burda & Sofroniew, 2014). However, it is unclear

whether human astrocytes exhibit such response patterns since most of our

knowledge about astrogliosis comes from murine systems. Currently,

divergent gene expression profiles found in mouse astrocytes were activated

by distinct stimuli (Hamby et al., 2012; Zamanian et al., 2012), while mouse

and human astrocytes did not share the same responses to

lipopolysaccharide (LPS) and IL-1 (Tarassishin, Suh, & Lee, 2014). These

results highlight interspecies differences regarding astrocytic responses.

Recently, murine astrocytes exposed to distinct activation stimuli were

classified in A1 and A2 according to their transcriptomic profile. Interestingly,

together with other cytokines, TNF-α was able to induce both profiles

(Liddelow et al., 2017). Indeed, TNF-α emerges as a key player on induction,

maintenance and profiling of astrogliosis.

Neural cells derived from induced pluripotent stem cells (iPSC) have

increasingly been used to model human diseases. In the last few years,

efforts have been made to improve differentiation protocols in order to obtain

and characterize human iPSC-derived astrocytes (Chandrasekaran, Avci,

Leist, Kobolak, & Dinnyes, 2016; Emdad, D'Souza, Kothari, Qadeer, &

Germano, 2012; Shaltouki, Peng, Liu, Rao, & Zeng, 2013; Tcw et al., 2017;


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Yan et al., 2013). Owing to the uniqueness of human astrocytes as well as

their responses to insults and inflammation, we evaluated human iPSC-

derived astrocytes regarding the canonical events associated with astrogliosis

in response to short- and long-term TNF-α stimuli as a mimetic

neuroinflammatory condition. These data were obtained following a

circumspect improvement of the protocol for obtaining human iPSC-derived

astrocytes described by Yan and colleagues, allowing us to show that these

cells present typical astroglial functional features (Yan et al., 2013). In order to

provide not only a characterization of human astrocyte-secreted proteins

following inflammatory stimulus, we also evaluated and characterized major

events related to the time course of astrogliosis such as i) TNF-α-mediated

nuclear translocation of NF-kB, ii) increases in secretion and gene expression

of inflammation-related cytokines, iii) alterations in the cell shape affecting the

aspect ratio and decreasing the area of astrocyte nuclei and iv) dysfunctional

aspartate/glutamate uptake. Although human iPSC-derived astrocytes have

been shown to respond to inflammatory stimuli (Perriot et al., 2018; Santos et

al., 2017), it was unclear whether human iPSC-derived astrocytes can

reproduce in vitro the temporally-ordered physiological changes typical of

astrogliopathology.

2. Material and Methods

2.1 Generation of human iPSC-derived astrocytes Human iPSC-derived astrocytes were differentiated from human iPSC-derived

neural stem cells (NSC) obtained from iPSC of four healthy subjects. Neural

stem cells (NSC) were obtained according to a protocol described previously


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(Yan et al., 2013). Three of these cell lines were used in other studies from

our research group (Casas et al., 2018; Garcez et al., 2017). One cell line was

obtained from a female subject (Subject 1 - available at Coriell Institute

Biobank with the name GM23279A) and the other three from male subjects

which cells were reprogrammed at the D’Or Institute for Research and

Education (Subjects 2, 3 and 4). Reprogramming of human cells was

approved by the ethics committee of Copa D’Or Hospital (CAAE number

60944916.5.0000.5249, approval number 1.791.182). Human cell

experiments were performed according to Copa D’Or Hospital regulation.

Table 1 exhibits summarized information regarding cell lines used in this

study. NSC were plated at density of 5 x104 cells/cm2 in 75 cm2 culture flasks,

pre-coated with Geltrex (A1413301 - ThermoFisher) in NSC expansion

medium containing 50% Advanced DMEM/F12 (12634-010 - Thermo Fisher

Scientific, MA, USA), 50% Neurobasal (21103-049 - Thermo Fisher Scientific,

MA, USA) and neural induction supplement (a16477-01 - Thermo Fisher

Scientific, MA, USA). On the following day, medium was replaced by astrocyte

induction medium (AIM) composed of DMEM/F12 (11330-032 – Thermo

Fisher Scientific, MA, USA), N2 supplement (17502001 – Thermo Fisher

Scientific, MA, USA) and 1% fetal bovine serum (FBS) (12657029 – Thermo

Fisher Scientific, MA, USA). AIM was changed every other day for 21 days.

During this period, when reaching confluence, cells were passed at a ratio of

1:4 using Accutase (A6964 – Sigma Aldrich, MO, USA) to new Geltrex-coated

flasks. AIM was changed on the day after cell passaging. By the end of the 21

days of differentiation, cells were exposed to astrocyte medium containing 10

% FBS in DMEM/F12. At this stage cells were named human iPSC-derived


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radial glia-like cells due to their positive labeling for radial glia markers PAX6

and phosphorylated Vimentin, which decrease with time in culture (Garcez et

al., 2017). From then on, after reaching confluence, cells were passed at a

ratio of 1:2 in the absence of Geltrex. Medium was changed twice a week and

on the following day after every cell passage. Human iPSC-derived radial glia-

like cells were kept in culture for additional four weeks in order to expand the

number of cells and to enhance maturing of astroglial functions. All

experiments described in this paper were performed from day 49 after the

beginning of astrocyte differentiation (Fig. 1a).

2.2 In vitro Astrogliosis

Human iPSC-derived astrocytes were seeded in non-coated multiwell plates

or 75 cm2 flasks at densities varying from 2 x 104 cells/cm2 to 6.25 x 103

cells/cm2. Astrocyte medium was changed at day 1 and 3 after each passage.

At day 4, astrocyte medium was replaced by serum deprived medium

(DMEM/F12) for 24 h and thereafter 1 - 50 ng/ml of human recombinant TNF-

α (717904 – BioLegend, CA, USA) or vehicle (DMEM/F12) were added

directly to the cells. Cells were then cultured for an additional 1 h, 24 h or 5

days and samples were collected/processed/analyzed according to the

experiment (Fig. 1b).

2.3 Immunocytochemistry and cell morphology analyses

Human iPSC-derived astrocytes were seeded at a density of 6.25 x 103

cells/cm2 on 96-multiwell Cell-Carrier plates (PerkinElmer, MA, USA). Four

days after plating, cells were exposed to serum-deprived medium and in the


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following day, concentrations of TNF-α ranging from 1 - 50 ng/ml were added.

For NF-kB translocation experiments, cells were fixed 1 h after TNF-α

exposure. For GFAP, Vimentin and morphology analyses, cells were fixed 5

days after TNF-α stimulus. Cells were fixed with 4 % paraformaldehyde

(Sigma Aldrich, MO, USA) in phosphate-buffered saline for 20 minutes,

permeabilized with 0.3% Triton X-100 (Sigma Aldrich, MO, USA) and then

exposed to blocking solution containing 2 % bovine serum albumin (Sigma

Aldrich, MO, USA). Next, an overnight incubation at 4oC with primary

antibodies was performed. We have used the following antibodies in this

study: mouse anti-NF-kB p65 (1:100; sc-8008 - Santa Cruz Biotechnology,

TX, USA), rabbit anti-Vimentin (1:2000; ab92547 – abcam, Cambridge, UK),

mouse anti-GFAP (1:200; MO15052 – Neuromics, MN, USA). Subsequently,

samples were incubated with the following secondary antibodies: goat anti-

rabbit Alexa Fluor 594 IgG (1:400; A-11037 - Thermo Fisher Scientific, MA,

USA) and goat anti-mouse Alexa Fluor 488 IgG (1:400; A-11001 - Thermo

Fisher Scientific, MA, USA) and goat anti-mouse Alexa Fluor 594 IgG (1:400;

A-11032 - Thermo Fisher Scientific, MA, USA). Nuclei were stained with 0.5

µg/mL 4′-6-diamino-2-phenylindole (DAPI) for 5 minutes. Images were

acquired with the Operetta high-content imaging system using 10 x and 20 x

objectives (PerkinElmer, MA, USA). Total number of cells was calculated by

DAPI stained nuclei counting. Densitometry and cell morphology analyses

were performed using software Harmony 5.1 (PerkinElmer, MA, USA). Eleven

different fields from triplicate wells per experimental condition were used for

quantification.


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2.4 Multiplex analysis of cytokines and BDNF

Cultures of human iPSC-derived astrocytes were seeded at a density of 2 x

104 cells/cm2 in 75 cm2 culture flasks. Four days after plating, cells were

exposed to 24 h of serum-deprived medium. Then, 10 ng/ml TNF-α was

added for an additional period of 24 h. After this incubation time, conditioned

medium was collected, aliquoted and immediately frozen and stored at -80oC.

ProcartaPlex™ bead-based multiplex immunoassays were performed for

simultaneous detection and quantitation of multiple protein targets in cell

culture supernatant. The platform MAGPIX™ was used for simultaneous

detection of BDNF, INF-γ, IL-1β, IL-10, IL-13, IL-2, IL-4, IL-6, IL-8 and TNF-α

in a single sample according to the manufacturer’s instructions. Briefly, 50

μl/well of coated magnetic beads solution (Luminex™ Corporation) were

added to a 96-well plate. The plate was inserted onto a magnetic plate

washer; beads were allowed to accumulate on the bottom of each well and

were then washed. Samples or standards (50 μl) were added to the wells and

then incubated at room temperature for two hours on a Capp 18-X multiwell

plate shaking platform (500 rpm). The plate was washed again twice and the

detection antibody mixture was added and incubated at room temperature for

30 minutes on a shaking platform (500 rpm). The streptavidin solution was

added to each well and incubated at room temperature for 30 minutes (500

rpm). After washing, reading buffer was added into each well and incubated at

room temperature for 5 minutes (500 rpm). Data were acquired on

MAGPIX™.

2.5 Quantitative Real time PCR


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Cultures of human iPSC-derived astrocytes were seeded at a density of 2x104

cells/cm2 in T-75 culture flasks. Four days after plating, cells were exposed to

serum-deprived medium. After 24 h of serum deprivation, 10 ng/ml TNF-α

was added for an additional period of 24 h. Cells were then detached with

Accutase (Merck, Darmstadt, Germany), centrifuged at 300 g for 5 minutes

after which the supernatant was discarded and the resulting pellet was

submitted to RNA extraction. Total RNA was isolated using TRIzol™ reagent,

according to the manufacturer’s instructions (Thermo Fisher Scientific, MA,

USA). The total RNA was resuspended in a final volume of 12 µl of Nuclease-

free water and quantified by absorbance at 260 nm in a spectrophotometer

(NanoDrop 2000, Thermo Fisher Scientific, MA, USA). One µg of total RNA

was reverse-transcribed in a 20 µl reaction volume using M-MLV (Thermo

Fisher Scientific, MA, USA), the generated cDNA was diluted 5x and a

quantitative PCR was performed using Eva™ Green PCR Mix (Biotium, CA,

USA) in the StepOne Plus Real-Time PCR Platform (Applied Biosystems, CA,

USA). RT-qPCR was carried out to detect transcripts of the following genes:

tumor necrosis factor α (TNF-α; forward: 5’-CTGCACTTTGGAGTGATCGG-

3’; reverse: 5’-TGAGGGTTTGCTACAACATGGG-3’); interleukin 1β (IL-1β;

forward: 5’-CACGATGCACCTGTACGATCA-3’; reverse: 5’-

GTTGCTCCATATCCTGTCCCT-3’); interleukin 6 (IL-6; forward: 5´-

TACCCCCAGGAGAAGATTCC-3´; reverse: 5´-

GCCATCTTTGGAAGGTTCAG-3´); glyceraldehyde-3-phosphate

dehydrogenase (GAPDH; forward: 5’-GCCCTCAACGACCACTTTG-3’;

reverse: 5’-CCACCACCCTGTTGCTGTAG-3’); hypoxanthine

phosphoribosyltransferase 1 (HPRT-1; forward 5´-


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CGTCGTGATTAGTGATGATGAACC-3´; reverse: 5´-

AGAGGGCTACAATGTGATGGC-3´); ribosomal protein lateral stalk subunit

P0 (RPLP0; forward: 5’-TTAAACCCTGCGTGGCAATC-3’; reverse: 5’-

ATCTGCTTGGAGCCCACATT-3’); and importin-8 (IPO8; forward: 5’-

TCCGAACTATTATCGACAGGACC-3’; reverse: 5’-

GTTCAAAGAGCCGAGCTACAA-3’). All primers used in this study were

validated by serial dilution to allow reaction efficiency calculation. Efficiencies

from all tested primers ranged from 90%-110%, and the maximum allowed

difference in efficiencies for primers used to estimate fold change of each

gene was 10% (data not show). RT-qPCR data was calculated by the 2-ΔCt

method using the geometric mean of RPLP0, GAPDH and IPO8 (IL-1β) or

GAPDH and HPRT1 (IL-6) to normalize results.

2.6 D-Aspartate uptake

Cultures of human iPSC-derived astrocytes were seeded at a density of 2x104

cells/cm2 on 24-well plates. Four days after plating, cells were exposed to

serum-deprived medium. After 24 h of serum deprivation, 10 ng/ml TNF-α

was added for an additional period of 24 h. Cultures were then incubated with

1 μCi/ml of 2,3-[3H]-D-aspartate (11.3 Ci/mmol) in Hanks saline solution

(HBSS) for 15, 30 and 60 minutes. The competitive inhibitor of the excitatory

amino acid transporters (EAATs) family, 100 μM DL-threo-β-

Benzyloxyaspartic acid (DL-TBOA), was added 10 minutes prior to 2,3-[3H]-D-

aspartate. After incubating periods, uptake was terminated by washing cells

with HBSS in order to remove non-incorporated 2,3-[3H]-D-aspartate, followed

by cell lysis. Aliquots were taken for intracellular radioactivity quantified by


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scintillation counting. Experiments were performed with 2 replicates per

experimental group in all time points analyzed and the average value of both

replicates was used for statistical analyses.

2.7 Cell death assay

Cell death was assessed 5 days after exposing cells to 10 ng/mL TNF-α

exposure. Cells were loaded with the fluorescent dye ethidium homodimer (2

µM - LIVE/DEAD Cell Imaging Kit, Thermo-Fisher, MA, USA) for 30 min. Live

cell images were acquired with the Operetta high-content imaging system

using a 20x objective (PerkinElmer, MA, USA). Cell death was determined by

the ratio of ethidium homodimer labeling per DAPI labeling. High-content

image analysis Harmony 5.1 was the software used for data analysis

(PerkinElmer, MA, USA). Seven different fields from duplicate wells per

experimental condition were used for quantification.

2.8 Statistical Analyses

Statistical comparisons between two experimental groups were performed by

Unpaired Student t‐test. NF-kB translocation experiments were analyzed by

One-way ANOVA followed by Tukey´s post hoc test. Aspartate uptake

experiments were analyzed by One-way ANOVA followed by Dunnett´s post

hoc test. Statistical significance was considered for P < 0.05. GraphPad

Prism v8.02 (GraphPad Software, CA, USA) was the software used for data

analyses and graphics.

3. Results


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3.1 TNF-α induces NF-kB nuclear translocation in iPSC-derived astrocytes

First, we aimed to determine whether human iPSC-derived astrocytes were

responsive to TNF-α by analyzing nuclear translocation of the transcription

regulator NF-kB, a mandatory step on astrogliosis induction. It can be noted

that cells incubated for 1 hour with TNF-α ranging from 1 to 50 ng/mL showed

nuclear translocation of NF-kB in all tested conditions (Fig. 2a). The increase

of NF-kB in the nucleus was concentration-dependent, with 1, 5, 10, 20 and

50 ng/ml TNF-α promoting increases of NF-kB immunoreactivity around 97%,

138%, 156%, 186% and 184%, respectively (Fig. 2b). When the whole cell

area was analyzed, a trend toward increasing was observed for NF-kB in all

concentrations of TNF-α tested, although it did not reach statistical

significance (Fig. 2c). The translocation index of NF-kB (calculated by

immunoreactivity of nuclei/cell area ratio), revealed that TNF-α induced a

significant increase in the NF-kB translocation index in all concentrations

tested. Similar to nuclei NF-kB immunoreactivity, the increase was dose

dependent, as 1, 5, 10, 20 and 50 ng/ml of TNF-α promoted an increase of

NF-kB nuclear translocation around 74%, 110%, 106%, 130% and 138%,

respectively (Fig. 2d). Despite the fact that cells were responsive to 1 ng/ml

TNF-α for NF-kB nuclei translocation, cells that received 10, 20 or 50 ng/ml

TNF-α were even more effective on recruiting NF-kB to the nucleus. Based on

these findings, 10 ng/mL TNF-a was chosen for further experiments in order

to promote astrocytes activation.

3.2 Inflammation-related cytokines are increased after TNF-α stimulation


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The release of cytokines is a hallmark of inflammatory response and also

astrogliosis. Therefore, in order to map the secretion of immunomodulators by

human iPSC-derived astrocytes activated by TNF-α, we performed a multiplex

analysis of cytokines using conditioned media from these cells. Based on the

literature, cytokines were classified into three major categories related to their

primary biological roles on inflammation: pro-inflammatory, modulatory or anti-

inflammatory, and also brain-derived neurotrophic factor (BDNF). The

secretion of pro-inflammatory cytokines IL-1β, IL-8 and IFN-γ was significantly

stimulated by TNF-α. While IL-1β secretion was increased in 425% (from

12.32 ± 1.11 to 64.72 ± 14.05 pg/mL) and 254% for IL-8 (from 1.09 ± 0.16 to

3.85 ± 0.39 ng/mL), IFN-γ presented 890% increase (from 47.9 ± 22.5 to

474.3 ± 147.8 pg/ml) when compared to conditioned media from vehicle-

treated cells (Fig. 3a). As expected, IL-1β, IFN-γ and TNF-α were barely

detectable in conditioned media of non-stimulated human iPSC-derived

astrocytes. TNF-α was highly detected in the conditioned media from

stimulated cells, probably due to its remaining content from the activation

stimulus.

Regarding modulatory cytokines, conditioned media obtained after TNF-α

stimulus presented a 352% increase for IL-2 (from 0.23 ± 0.05 to 1.05 ± 0.14

ng/mL), 704% for IL-4 (from 0.24 ± 0.11 to 1.92 ± 0.16 pg/mL) and 311% for

IL-6 secretion (from 1.62 ± 0.59 to 6.67 ± 1.55 pg/ml) (Fig. 3b). Among anti-

inflammatory cytokines, while IL-10 levels were unchanged, secretion of IL-13

increased 618% (from 11.3 ± 7.4 to 81.4 ± 21.5 pg/mL) and BDNF increased

106% in the conditioned medium after TNF-α exposure (from 255.5 ± 41.4 to

527.5 ± 40.6 pg/mL) (Fig. 3c).


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3.3 Inflammation-related cytokines display increased expression after TNF-α

stimulation

Despite the fact that most cytokines have short half-life, increases in their

secretion should impact gene expression and continuous production was

expected. Thus, quantitative real time PCR (qPCR) was conducted to verify

the expression of cytokines, which secretion was found to be increased in the

conditioned medium (Fig. 4). Indeed, both IL-6 and IL-1β genes expression

showed ~15.6- and ~ 6.0-fold increase, respectively, following TNF-α

exposure (Fig. 4a,b). In addition, TNF-α expression was detected only in

stimulated cells, but not in any replicates within the control group (Fig. 4c). In

order to show that control samples were viable in terms of gene expression,

we also analyzed the expression levels of housekeeping genes RPLP0,

GAPDH and IPO8 from the same samples used in Figures 4b and 4c. No

differences were found among experimental groups (Supplementary Figure

1).

3.4 TNF-α induced morphological alterations and upregulation of intermediate

filaments Vimentin and GFAP

The incubation of astrocytes with 10 ng/ml TNF-α during five consecutive

days (in serum-free conditions) triggered typical phenotypes of astrogliosis, as

revealed by increased immunoreactivity for Vimentin and GFAP (Fig. 5a, b, c)

as well as shrunken nuclei (Fig. 5f). The incubation with 10 ng/ml TNF-α did

not change measurements of cell area (Fig. 5c). However, some vimentin-

stained astrocytes clearly showed a thin, process-devoid and polarized


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phenotype after treatment with 10 ng/ml TNF-α. To identify these cells, we

calculated the aspect ratio (length to width ratio) from vimentin

immunoreactivity. A greater length/width ratio would indicate cells with

relatively thin shapes (Fig. 5f). We confirmed that TNF-α promotes an

increase in cell polarization (25%), which is typically found under inflammatory

conditions (Jang et al., 2013).

3.5 Glutamate uptake is impaired in human iPSC-derived astrocytes exposed

to TNF-α

One important functional feature of astrogliosis is the dysfunction of

glutamate/aspartate transporters. In order to accurately measure uptake

activity, the non–metabolized analog D-[3H] Aspartate was used. Aspartate

uptake was carried out 1 and 5 days after TNF-α incubation. At both time

points, TNF-α was able to inhibit D-[3H]aspartate uptake by human iPSC-

derived astrocytes (Fig. 6a,b). At 60 minutes of aspartate uptake, a reduction

of ~47% was found in cells exposed to TNF-α for 1 day (Fig. 6a). When cells

were exposed to TNF-α for 5 days, a reduction in aspartate uptake was

observed at 5, 15, 30- and 60-minutes time points (61.5%, 72.4%, 73.3% and

51.5%, respectively) when compared to control cells (Fig. 6b). Also, human

iPSC-derived astrocytes displayed a highly specific aspartate uptake activity,

as evidenced by full inhibition with the competitive inhibitor of excitatory amino

acid transporters DL-TBOA (Fig. 6a, b). In the presence of DL-TBOA, TNF-α

had no effect on transporters activity (Fig. 6a, b). Since the astrogliosis

induction was carried out in the absence of serum, we assessed astrocyte

viability after five consecutive days of TNF-α exposure. The removal of fetal


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bovine serum from culture medium induced a small increment in cell death:

5% of cells incorporated ethidium bromide while only 0.7% were ethidium-

positive among serum-supplemented cells (Fig. 6c). Similarly, TNF-α in the

absence of serum decreased astrocyte viability in 7.4%. No significant

difference in cell death was observed between cells exposed to TNF-α and

vehicle (Fig. 6c).

4. Discussion

In this study, human iPSC-derived astrocytes were characterized with regard

to their responsiveness to short and long-term inflammatory conditions. Thus,

human iPSC-derived astrocytes from healthy subjects were challenged with

the pro-inflammatory cytokine TNF-α, which is able to initiate and sustain

long-term features of astrogliosis (Cui, Huang, Tian, Zhao, & Zheng, 2011).

In glial cells, NF-kB remains in its physiologically latent cytoplasmic form,

being translocated to the nucleus under inflammation (Kaltschmidt &

Kaltschmidt, 2009). We noticed that human iPSC-derived astrocytes were

capable of responding to short term TNF-α exposure through the translocation

of NF-kB to the nucleus, a predicted event that occurs within an hour after

inflammatory stimuli (Nelson et al., 2004; Romano, Freudenthal, Merlo, &

Routtenberg, 2006).

Secretome analysis revealed that high levels of TNF-α were detected in the

conditioned media 24 h after exposing cells to this cytokine, but it was difficult

to distinguish between residual levels arising from the stimulus or secretion.

However, TNF-α was almost undetectable in the conditioned medium and cell

extracts from non-stimulated astrocytes, confirming that TNF-α is essentially


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produced by stimulated astrocytes. Since TNF-α via NF-kB activation triggers

inflammatory events such as cytokines production, we also found that upon

short-term TNF-α stimulation (24 h), either modulatory cytokines IL-2, IL-4

and IL-6 or the chemotactic and inflammatory cytokine IL-8 had their levels

increased from pg/mL to ng/ml range, which is typical for inflammatory

responses or pathological conditions (Stenken & Poschenrieder, 2015).

However, IFN-γ and BDNF were also increased in the conditioned medium

from astrocytes exposed to TNF-α, but to a lesser extent. It is important to

take into account that over the course of acute inflammatory stimuli many

factors and cytokines are expressed in a coordinated manner. Since IFN-γ is

a pro-inflammatory factor, increases in its secretion may be important to

induce and sustain astrogliosis (Yong et al., 1991). In a previous report,

cultured rat astrocytes were exposed to different inflammatory stimuli, but only

TNF-α was able to increase expression and secretion of BDNF via NF-kB

activation (Saha, Liu, & Pahan, 2006). Even though the activity-dependent

secretion BDNF is well documented in neuronal cells, the secretory nature of

astrocytes suggests that increased BDNF secretion by TNF-α might be an

attempt of stimulated-astrocytes to provide a neurotrophic factor that will

rescue viability following acute inflammatory stimuli.

Interestingly, relatively high levels for IL-6 and IL-8 were detected in the

conditioned medium from non-stimulated astrocytes (1.6 and 1.0 ng/ml,

respectively). Of note, astrocytes are a major source of IL-6, which is

important for synapse formation (Wei et al., 2011), maturation of dendritic

spines (Wei et al., 2012) and sprouting of glutamatergic connections

(Menezes et al., 2016), while IL-8 regulates angiogenesis by enhancing


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survival and proliferation of endothelial cells (Li, Dubey, Varney, Dave, &

Singh, 2003).

As expected for mature astrocytes, almost all human iPSC-derived astrocytes

were immunoreactive for GFAP and vimentin, though not all astrocytes

necessarily express GFAP in the human brain tissue (Kettenmann &

Verkhratsky, 2011; Kimelberg, 2004). Both GFAP and vimentin are

considered markers of astrocytes and their overexpression characterizes

primary features of reactive astrogliosis, since their deletion reduces reactive

gliosis and promotes synaptic regeneration (Wilhelmsson et al., 2004). TNF-α

is fairly known to induce overexpression of GFAP and vimentin (Perriot et al.,

2018; Zhang et al., 2016) and our human iPSC-derived astrocytes were

responsive to long-term TNF-α exposure by increasing GFAP and vimentin.

The long-term exposure to TNF-α was also able to alter astrocyte morphology

in vitro, since they displayed an elongated shape, namely polarized

astrocytes, resembling those in response to a wound (Peng & Carbonetto,

2012) or recruited to mold the glial scar around injured sites (Adams & Gallo,

2018).

It is well documented the crucial role of astrocytes in maintaining glutamate

homeostasis by continuous removal of extracellular glutamate from the

synaptic cleft by glutamate transporters, a mechanism that avoids glutamate

excitotoxicity (Rothstein et al., 1996). Recent evidences have shown that

extracellular glutamate stimulates glutamate release from astrocytes in order

to coordinate neuronal activity, pointing to an emerging role for astrocytes in

modulating both glutamate uptake and release and extending their therapeutic

potential (Mahmoud, Gharagozloo, Simard, & Gris, 2019). It could be noted


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that iPSC-derived astrocytes exposed to short term TNF-α had already

showed impairment in the aspartate uptake. As astrogliosis progresses due to

long term exposure to TNF-α, the aspartate uptake impairment worsened.

Importantly, the impairment of glutamate uptake by prolonged exposure to

TNF-α was not related to astrocyte viability, which confirms that they were

dysfunctional indeed. Since atrophic astrocytes have been proposed to be

dysfunctional, the impairment of glutamate uptake triggered by TNF-α

intertwines both events in the context of astrocytopathy (Pekny & Pekna,

2016). Astrogliosis is considered a complex response, which varies from

hypertrophy, atrophy, alteration in astrocytes biomarkers and dysfunction. Our

findings show that human iPSC-derived astrocytes can be functionally

activated, mimicking previously reported physiological aspects of astrogliosis

described in animal models, human primary cultures and post-mortem brain

studies. Astrogliopathology comprises both reactive astrogliosis and

astrocytopathy that can co-exist after an insult by a series of changes that

appear to be context- and disease-driven (Kim, Healey, Sepulveda-Orengo, &

Reissner, 2018; Pekny & Pekna, 2016). These changes vary over time with a

continuum of progressive alterations that can result in either beneficial or

detrimental effects on the surrounding tissue (Burda & Sofroniew, 2014).

The inflammatory processes share similarities and differences between

animal and human models, causing intense debate over the theme (Cauwels,

Vandendriessche, & Brouckaert, 2013; Warren et al., 2015). For example,

genomic responses to inflammatory challenges are divergent between both

species, although the same dataset has been subject of contradictory

interpretations in two different studies (Kodamullil et al., 2017; Mestas &


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22

Hughes, 2004; Seok et al., 2013; Takao & Miyakawa, 2015). Besides,

signaling pathways of neuroinflammation share similarities between rodents

and humans, but substantial differences were found for molecular and cellular

interactions (Kodamullil et al., 2017). Astrogliosis has been systematically

related to several CNS pathologies and the implementation of functional in

vitro models of human neural cells has been subject of research in the last

years. Therefore, a complete screening of inflammatory responses to different

stressing stimuli by human astrocytes may shed light in the pathophysiology

of neurodegenerative and psychiatric diseases. For instance, human iPSC-

based model of astrogliosis may improve the understanding of

neuroinflammation-related diseases and promote the development of new

therapeutic strategies.

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Tables

Table 1 – Cell lines used in the study.

Figure Legends

Figure 1 - Experimental Design. (a) Derivation of human iPSC-astrocytes.

Human neural stem cells (NSC) were differentiated from induced pluripotent

stem cells (iPSC) and they were exposed to astrocyte induction medium

Cell Line GenderAge of

collectionPrimary Cells Institution Previous Publications Use in Experiments

Subject 1 Female 20 Dermal Fibroblasts Coriell Biobank Casas et al., 2018 Fig. 2-6 & Suplementary Fig. 1

Subject 2 Male 37 Dermal FibroblastsD'Or Institute for

Research and EducationCasas et al., 2018 Fig. 2-6 & Suplementary Fig. 1

Subject 3 Male 31 Dermal FibroblastsD'Or Institute for

Research and EducationCasas et al., 2018 Garcez et al., 2017

Fig. 2-6 & Suplementary Fig. 1

Subject 4 Female 16 Urine cellsD'Or Institute for

Research and EducationNone Fig. 3-5 & Suplementary Fig. 1


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(AIM). After 21 days in AIM human iPSC-derived radial glia-like cells (RCG)

exhibited high proliferation rates and strong labeling for neural precursor

markers. After exposing RGC to astrocyte medium for additional 4 weeks,

human iPSC-derived astrocytes were obtained and used in the experiments.

(b) Induction of in vitro astrogliosis. Human iPSC-derived astrocytes were

exposed to serum-deprived culture medium for 24 h; then TNF-α was added

to the cells in order to analyze the canonical events over the course of

astrogliosis, as follows: i) NF-kB nuclear translocation was assessed 1 h after

TNF-α exposure; ii) expression and secretion of cytokines and D-aspartate

uptake assay were performed 24 h following TNF-α exposure; iii)

densitometry of the intermediate filaments, vimentin and GFAP, cell

morphology and viability, D-aspartate uptake assays were performed 5 days

after TNF-α stimulation in order to investigate chronic stage of astrogliosis.

Figure 2 – TNF-α-induced NF-kB nuclear translocation in human iPSC-

derived astrocytes. (a) - Photomicrographs of NF-kB immunostaining 1 h after

exposing cells to vehicle or five different concentrations of TNF-α.

Quantification of NF-kB immunoreactivity in both (b) – Nuclei and (c) – Whole

cell area, which were expressed in arbitrary units of immunofluorescence

(A.U.). (d) – The NF-kB translocation index (nuclei/whole cell area ratio). Data

are presented as means ± SEM of experiments performed in triplicates from 3

cell lines. *P < 0.01, different from vehicle; **P < 0.01, different from vehicle

and 1 ng/mL TNF- α. ***

P < 0.01, different from 10 ng/mL TNF-α. One-way

ANOVA followed Tukey´s post hoc test. Magnification: 100 x. Calibration bar:

200 μm.


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Figure 3 – Cytokines and BDNF secretion from human iPSC-derived

astrocytes. Conditioned media were collected after stimulating cells during 24

h with 10 ng/mL TNF-α. Cytokines and BDNF secretion was measured in the

conditioned media and compared with cells treated with vehicle. (a) - Pro-

inflammatory cytokines: Interleukin-1 beta (IL-1β), Interleukin-8 (IL-8),

Interferon gamma (IFN-γ) and Tumor necrosis factor alpha (TNF-α); (b) -

modulatory cytokines: Interleukin-2 (IL-2), Interleukin-4 (IL-4) and Interleukin-6

(IL-6); (c) - anti-inflammatory cytokines Interleukin-10 (IL-10), Interleukin-13

(IL-13) and Brain-derived neurotrophic factor (BDNF). Data are presented as

means ± SEM of concentrations in (pg/ml) of secreted factors. Conditioned

media were collected from 4 cell lines and the experiments were performed in

duplicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns - non-

significant. Unpaired Student´s t-test.

Figure 4 – Cytokines expression in cell extracts from human iPSC-derived

astrocytes. (a) – Interleukin-6; (b) – Interleukin-1 beta and (c) – TNF-α

expression was assessed 24 h after exposing cells to vehicle or 10 ng/mL

TNF-α. Data are presented as means ± SEM of fold change for (a) and (b)

and ΔCt (c) since the basal levels of TNF-α were below the limit of

quantification in non-stimulated cells. Experiments were performed in

triplicates from 4 cell lines. *P < 0.05; ****P < 0.0001; N.D. non detected.

Unpaired Student´s t-test.

Figure 5 – Morphological analysis of human iPSC-derived activated

astrocytes following 5 days TNF-α stimulation. (a) - Photomicrographs of


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human iPSC-derived astrocytes immunostained for vimentin (red), GFAP

(green) and DAPI (blue); (b) – Quantification of vimentin immunostaining; (c)

– Quantification of cell area in vimentin-stained cells. (d) – Percentage of

astrocyte polarization, which cells were classified according to increase of

length/width ratio of vimentin-stained labeling. (e) - Quantification of GFAP

immunostaining expressed in arbitrary units of immunofluorescence (A.U); (f)

- Quantification of DAPI-stained nuclei areas expressed as percentage of

micrometers. Data are presented as means ± SEM from 4 cell lines in

experiments performed in triplicates. **P < 0.01. Unpaired Student´s t-test.

Photomicrographs magnification: 200x. Calibration bar: 100 μm.

Figure 6 – Impairment of [3H] D-aspartate uptake by TNF-α in human iPSC-

derived astrocytes. Aspartate uptake was carried out (a) – 1 day or (b) – 5

days after exposing cells to vehicle or 10 ng/mL TNF-α. The competitive

inhibitor of glutamate transporters DL-TBOA was added 10 minutes prior to

aspartate. Data are presented as means ± SEM of the percentage of counts

per minute (cpm). (c) – Cell viability was evaluated by ethidium incorporation.

As a positive control for cell death, cells were lysed with Triton 2 %. As a

positive control for cell viability, cells grown in DMEM/F12 with 10 % SFB

were also evaluated. Data are presented as means ± SEM of the percentage

of ethidium incorporation (arbitrary units of fluorescence). Data from 3 cell

lines and experiments were performed in duplicates (a), (b) and

quadruplicates (c). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; One-

way ANOVA followed by Dunnett´s post hoc test. ns – Non-significant.


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Supplementary Figure 1 - Expression of housekeeping genes RPLP0,

GAPDH and IPO8 was not affected by TNF-α exposure in human iPSC-

derived astrocytes. Samples used in these experiments were also used in

data presented on Figures 4b and 4c. Results were normalized by average

values of all three housekeeping genes. Graphs represent data from 4 cell

lines in experiments performed in triplicates. ns non-significant. Data are

presented as means ± SEM.

Author Contributions

P.T. improved the protocol for obtaining and culturing of human iPSC-derived

astrocytes, performed cell morphology analyses and experiments for

astrogliosis characterization. P. T. and E. C. L. cultured and adjusted human

iPSC-derived astrocytes for all performed experiments. P.T., J. A. S. and I. M.

O. designed and analyzed NF-kB experiments. J. G., C. T. R, J. C. F. M and

D. P. G., performed and analyzed secretome data. P. L. C., S. D. and F. M. R.

performed and analyzed quantitative real time PCR experiments. P.T., E.C.L.

and A. L. M. V. conducted D-aspartate uptake assay and analysis. P. F. L.

and P. L. C. designed schematic figures. P.T. and S. K. R. designed the

research. P.T., P.F.L, J.A.S., L.O.P. and S.K.R. analyzed data, discussed

results and wrote the paper. All authors discussed results and validation

steps.


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TNF-α stimulation

Serum Starvation24h

TNF-α

iPSC-derivedAstrocytes

iPSC-derivedAstrocytes

NF-kB Translocation

24h:

iPSC-derivedNSC

iPSC-derivedRGC

GFAP & Vimentin DensitometryCell Morphology AnalysisD-Aspartate UptakeCell Death Assay

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(a)

Multiplex Analysis of CytokinesCytokines ExpressionD-Aspartate Uptake


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10 ng/ml TNF-α 20 ng/ml TNF-α 50 ng/ml TNF-α

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running title authors - biorxiv...2019/08/01 · ventura & harris, 1999). astrocytes are also...

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